PROJECT 5: OPTIMIZATION OF MART-1 TCR GENE TRANSFER FOR ANTI-MELANOMA IMMUNITY ABSTRACT TCR gene transfer is a feasible approach for treating metastatic melanoma and greatly expands the scope of adoptive transfer for cancer immunotherapy. In light of the low clinical response rate to standard therapies, further optimization of this therapeutic modality is urgently needed. Our long-term goal is to optimize the components involved in TCR gene transfer so that this treatment can be translated into a reliable therapeutic modality. Our preliminary studies have identified a high affinity MART-1 TCR from a patient with an unusually high population of high affinity MART-1 specific T cells. The experimental focus of this proposal is to evaluate various strategies to improve TCR gene transfer. The first specific aim is to optimize the MART-1 TCR for an enhanced anti-melanoma response. Success of this aim will enable us to overcome the intrinsic problem of TCR mispairing and obtain an engineered MART-1 TCR with improved affinity. The second specific aim is to optimize T cells that have been modified by a MART-1 TCR to yield an enhanced anti-melanoma response, which will allow us to validate the proposed genetic modification strategies for building better T cells and develop a novel means to generate optimal MART-1 T cells in vitro for adoptive transfer. The third specific aim is to optimize a lentiviral delivery system for TCR gene transfer. Accomplishment of this aim will provide us with new versions of lentiviral vectors with superior abilities to genetically modify mature T cells, hematopoietic stem cells and embryonic stem cells. Taken together, these novel studies will contribute significantly to the further success of the next wave of investigation into TCR gene transfer.